Cinnamoyl-piperazine derivatives and their use as par-1 antagonists

ABSTRACT

The present invention relates to compounds of general formula (I): 
     
       
         
         
             
             
         
       
     
     wherein:
 
R 1  represents:
         halogen, CN or NO 2 ;
 
R 2  represents:
   hydrogen or halogen;
 
n represents:
   1 or 2;
 
R 3  represents:
   phenyl substituted by one or more halogens or C 1 -C 6  alkyls; or a cyclohexyl;
 
as well as the therapeutically-acceptable salts or solvates thereof.
       

     These compounds are useful as protease-activated receptor-1 (PAR-1) antagonists, particularly in the treatment of thrombosis.

The present invention relates to cinnamoyl-piperazine derivatives, amethod of manufacturing same, pharmaceutical compositions comprised ofsame and the use of same as drugs for the treatment and/or prevention ofarterial and/or venous thrombosis, acute coronary syndromes, restenosis,stable angina, heart rhythm disorders, myocardial infarction,hypertension, heart failure, stroke, inflammatory disorders, pulmonarydiseases, gastrointestinal diseases, fibrosis development in chronicliver disease patients, cancer and skin diseases. The present inventionalso relates to combinations of the inventive compounds with othercardiovascular agents.

Thrombosis is regarded as a primary factor in vascular occlusion, whichis the cause of a number of pathophysiological complications.Antithrombotic therapy is thus extremely important as it can reduce therisk of cardiovascular mortality and coronary events. Although severaltypes of molecules have shown effective antithrombotic activity in man,a need for novel molecules remains. Indeed, improvements can be made toexisting compounds, some of which have a negative impact on bleedingtime or are accompanied by other undesirable side effects (such as, forexample, the risk of ulcer with aspirin).

Protease-activated receptor-1 (PAR-1) was recently cloned (Vu et al.,Cell, 1991, 64: 1057-1068) and its mechanism of action elucidated(Coughlin et al., J. Clin. Invest. 1992, 89(2): 351-355). This receptor,notably present on the surface of platelets but also on the surface ofendothelial cells (O'Brien et al., J. Biol. Chem. 2000, 275:13502-13509), smooth muscle cells (Hamilton et al., Br. J. Pharmacol.2000, 130: 181-188) and fibroblasts (Hung et al., J. Cell. Biol. 1992,116(3): 827-832), is activated by thrombin and thus is also calledthrombin receptor. The N-terminus of the protein is cleaved by thrombinbetween arginine 41 and serine 42 to free a new end which will act,after folding upon the active site, as a receptor agonist (Vu et al.,Nature, 1991, 353, 674-677). With respect to platelets, this specificPAR-1 receptor activation mechanism leads to thrombin-mediated plateletaggregation.

The blocking of this activation, for example with PAR-1 receptorantagonists, can inhibit thrombin-mediated platelet aggregation (Ahn etal., Drug of the Future, 2001, 26: 1065-1085). The blocking of thesereceptors can thus lead to the treatment or prevention of thrombosis(Derian et al., J. Pharmacol. Exp. Ther., 2003, 855-861), acute coronarysyndromes (Ossovskaya et al., Physiol. Rev., 2004, 84: 579-621) andrestenosis (Maryanoff et al., Curr. Med. Chem. Cardiovasc. Hematol.Agents., 2003, 13-36) and can reduce myocardial necroses duringinfarction or reperfusion (Steinberg et al., Mol. Pharmacol. 2005, 67:2-11). PAR-1 antagonist activity can prevent certain inflammatorydiseases in the pulmonary system (Moffatt et al., Curr. Op. Pharmacol.,2004, 221-229) and in the gastrointestinal system (Vergnolle et al., J.Clin. Invest., 2004, 1444-1456). PAR-1 antagonists can also be of use inthe treatment of fibroses in patients with chronic liver disease(Fiorucci et al., Hepatology, 2004, 39: 365-375). They can also be ofuse as anti-cancer agents given that they act to control cellularproliferation and metastases (Evan-Ram et al., Nat. Med., 1998, 909-914;Boire et al., Cell., 2005, 120: 303-313). Lastly, PAR-1 antagonists canbe of interest in dermatology to treat certain skin diseases (Schechteret al., J. Cell. Physiol., 1998, 176:365-373; Algermissen et al., Arch.Dermatol. Res., 2000, 292:488-495; Meyer-Hoffert et al., Exp. Dermatol.,2004, 13: 234-241).

The present invention relates to a novel class of PAR-1 antagonists thatare distinguished from the prior art by their different chemicalstructure and their remarkable biological property.

Compounds of the present invention are of general formula (I):

wherein:R₁ represents:

-   -   halogen, CN or NO₂;        R₂ represents:    -   hydrogen or halogen;        n represents:    -   1 or 2;        R₃ represents:    -   phenyl substituted by one or more halogens or C₁-C₆ alkyls; or a        cyclohexyl;        or the therapeutically-acceptable salts or solvates thereof.

In the preceding definitions:

All combinations of substituents or variables are possible insofar asthey lead to stable compounds.

The term “halogen” represents fluorine, chlorine, bromine or iodine.

The term “alkyl” represents linear or branched, saturated or unsaturatedaliphatic hydrocarbon chains comprising the specified number of carbonatoms.

Therapeutically-acceptable salts of compounds of the present inventioninclude conventional nontoxic salts of compounds of the invention suchas those formed from organic or inorganic acids. As an example, thefollowing can be cited: inorganic acid salts such as hydrochloric,hydrobromic, phosphoric and sulfuric acids, as well as organic acidsalts such as acetic, trifluoroacetic, propionic, succinic, fumaric,malic, tartaric, citric, ascorbic, maleic, glutamic, benzoic, salicylic,toluenesulfonic, methanesulfonic, stearic and lactic acids.

These salts can be synthesized from compounds of the inventioncontaining a base moiety and corresponding acids according toconventional chemical methods.

Therapeutically-acceptable solvates of compounds of the presentinvention include conventional solvates such as those formed during thefinal preparation step of compounds of the invention as a result of thepresence of solvents. Solvates due to the presence of water or ethanolcan be cited as an example.

Among the compounds of general formula (I) according to the presentinvention, one particularly advantageous class of compounds arecompounds of general formula (I) wherein R₁ is halogen, R₂ is hydrogen,n equals 1 and R₃ is phenyl substituted by one or more halogens or C₁-C₆alkyls.

Another particularly advantageous class of compounds according to thepresent invention is compounds of general formula (I) wherein R₁ iscyano, R₂ is hydrogen, n equals 1 and R₃ is phenyl substituted by one ormore halogens or C₁-C₆ alkyls.

Another particularly advantageous class of compounds according to thepresent invention is compounds of general formula (t) wherein R₁ ishalogen, R₂ is hydrogen, n equals 1 and R₃ is cyclohexyl.

Another particularly advantageous class of compounds according to thepresent invention is compounds of general formula (I) wherein R₁ iscyano, R₂ is hydrogen, n equals 1 and R₃ is cyclohexyl.

The present invention also relates to the preparation of compounds ofgeneral formula (I) by the general methods described in the followingsynthesis diagrams supplemented by, as the case may be, any standardtechnique described in the literature, known to those persons skilled inthe art, or presented in the experiments section.

Diagram 1 illustrates the first general method that can be used for thepreparation of compounds of general formula (I). In the general formulasabove, R₁, R₂, R₃ and n are defined as in the preceding description ofgeneral formula (I). P₁ represents a protective group. X can represent aleaving group such as chlorine. In this case, the first step consists ofthe reaction between an acid chloride and an amine. This reaction can becarried out by methods and techniques known to those persons skilled inthe art. A particularly advantageous method consists of reacting the twoentities in the presence of an organic or inorganic base such as, forexample, Et₃N, iPr₂NEt, pyridine, NaH, Cs₂CO₃ or K₂CO₃ in a solvent suchas THF, dichloromethane, DMF or DMSO at a temperature between −20° and100° C. X can also represent hydroxyl. In this case, the first step is acondensation reaction between the carboxylic acid (II) and the amine(III). This reaction can be carried out by methods and techniques knownto those persons skilled in the art. A particularly advantageous methodconsists of reacting these two entities in the presence of 1-(3dimethylaminopropyl)-3-ethyl-carbodiimide (EDC),3-hydroxy-1,2,3-benzotriazin-4(3H)-one, and a tertiary amine such asdiisopropylethylamine in a polar aprotic solvent, such asdichloromethane or DMF, at a temperature between −15° C. and 40° C.

After deprotection of the intermediate (IV) by methods and techniquesknown to those skilled in the art (“Protective Groups in OrganicSynthesis,” T. W. Greene, John Wiley & Sons, 1981 and “ProtectingGroups,” P. J. Kocienski, Thieme Verlag, 1994), the intermediateobtained can react with a reagent of formula R₃(CH₂)_(n)Y, wherein Yrepresents a leaving group such as, for example, Cl, Br, I, OSO₂CH₃,OSO₂CF₃ or O-tosyl. In this case, the reaction will be carried out inthe presence of an organic or inorganic base such as, for example, Et₃N,iPr₂NEt, NaH, Cs₂CO₃ or K₂CO₃ capable of being supported on a resin suchas PS-DIEA or MP-carbonate, in a polar anhydrous solvent such asdichloromethane, THF, DMF or DMSO at a temperature between −20° and 100°C. Another preparation method consists of carrying out a reducingamination reaction using an aldehyde of formula R₃—(CH₂)_(n-1)—CHO inwhich R₃ and n are as defined previously, with the deprotected amine ofgeneral formula (IV) and a reducing agent such as NaBH₄, NaBH₃CN orNaBH(OAc)₃ capable of being supported on a resin such as MP—BH₃CN, in apolar solvent such as 1,2-dichloroethane, dichloromethane, THF, DMF orMeOH, at a pH that can be controlled by the addition of an acid such asacetic acid, at a temperature between −20° C. and 100° C.

Diagram 2 illustrates the second general method that can be used for thepreparation of compounds of general formula (I). In the general formulasabove, R₁, R₂, R₃ and n are defined as in the preceding description ofgeneral formula (I). X can represent a leaving group such as chlorine.In this case, synthesis consists of the reaction between an acidchloride and an amine. This reaction can be carried out by methods andtechniques known to those persons skilled in the art. A particularlyadvantageous method consists of reacting the two entities in thepresence of an organic or inorganic base such as, for example, Et₃N,iPr₂NEt, pyridine, NaH, Cs₂CO₃ or K₂CO₃ in a solvent such as THF,dichloromethane, DMF or DMSO at a temperature between −20° and 100° C.

X can also represent hydroxyl. In this case, synthesis consists ofcondensation between the carboxylic acid (II) and the amine (V). Thereaction can be carried out by methods and techniques known to thosepersons skilled in the art. A particularly advantageous method consistsof condensing a carboxylic acid of general formula (II) with an amine ofgeneral formula (V) in the presence of1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide (EDC),3-hydroxy-1,2,3-benzotriazin-4(3H)-one and a tertiary amine such asdiisopropylethylamine, in a polar aprotic solvent such asdichloromethane, at a temperature between −15° C. and 40° C.

When it is desired to isolate a compound of general formula (I)containing at least one base function in salt state by the addition ofan acid, such a result can be achieved by treating the free base ofgeneral formula (I) (in which at least one base function is present)with a suitable acid, preferably in an equivalent quantity.

The examples which follow illustrate the invention without limiting itsscope in any way.

Example 13-(2-Chloro-phenyl)-1-[4-(4-fluoro-benzyl)-piperazin-1-yl]-propenone

Example 1A -4-(4-Fluoro-benzyl)-piperazine-1-carboxylic Acid tert-butylEster

Piperazine-1-carboxylic acid tert-butyl ester (5.0 g, 26.8 mmol) insolution in dichloromethane (100 ml) in the presence ofdiisopropylethylamine (DIEA) (5.59 ml, 40.2 mmol) is treated with4-fluorobenzyl bromide (3.68 ml, 29.5 mmol) at room temperature. After16 hours of agitation the reaction mixture is diluted withdichloromethane and washed with water. The organic phase is dried onMgSO₄, filtered and evaporated to dryness. The syrup obtained ispurified by silica column chromatography and eluted with a 98/2 to 95/5CH₂Cl₂/MeOH mixture. Product 1A is isolated in the form of a white solid(7.03 g, 88%).

¹H NMR, DMSO-d₆ (ppm): 1.38 (s, 9H); 2.29 (t, 4H); 3.30 (broad s, 4H);3.45 (s, 2H); 7.14 (t, 2H); 7.32 (dd, 2H).

Example 1B -4-(4-Fluoro-benzyl)-piperazine

4-(4-Fluoro-benzyl)-piperazine-1-carboxylic acid tert-butyl ester (7.03g, 23.8 mmol) in solution in toluene (300 ml) is treated withtrifluoroacetic acid (53.2 ml, 716 mmol) at room temperature. After 2hours of agitation the reaction mixture is diluted with dichloromethane,washed with 1 N soda and then with water. The organic phase is dried onMgSO₄, filtered and evaporated to dryness. The crude product is isolatedfor the following reaction (4.2 g, 90%).

Example 1-3-(2-Chloro-phenyl)-1-[4-(4-fluoro-benzyl)-piperazin-1-yl]-propenone

A mixture of 2-chloro-cinnamic acid (2.43 g, 13.3 mmol) and4-(4-fluoro-benzyl)-piperazine (2.16 g, 11.1 mmol) in solution indichloromethane (70 ml) is treated with1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) hydrochloride (2.55g, 13.3 mmol) and 3-hydroxy-1,2,3-benzotriazin-4(3H)-one (HOOBT) (2.17g, 13.3 mmol) in the presence of DIEA (3.86 ml, 22.2 mmol) at roomtemperature. After 48 hours of agitation the reaction mixture is dilutedwith ethyl acetate and washed with 1 N soda and then with water. Theorganic phase is dried on MgSO₄, filtered and evaporated to dryness. Thesyrup obtained is purified by silica column chromatography and elutedwith a 97.75/2/0.25 CH₂Cl₂/MeOH/NH₄OH mixture. Product 1 is isolated inthe form of a yellow oil (3.77 g, 95%). This product is taken up withethyl acetate and salified by addition of a solution of HCl in ether toyield the corresponding hydrochloride in the form of a yellow solid(4.14 g)

¹H NMR, DMSO-d₆ (ppm): 3.02 (m, 2H); 3.21 (t, 1H); 3.63 (t, 1H); 4.05(broad s, 2H); 4.34 (s, 2H); 4.52 (t, 2H); 7.32 (m, 3H); 7.43 (m, 2H);7.53 (m, 1H); 7.66 (m, 2H); 7.92 (d, 1H); 8.00 (m, 1H); 11.49 (s, 1H).

Mass spectrum (ESI+): m/z 359 (M+H⁺)

Elemental analysis: C₂₀H₂₀N₂O₁·HCl and 0.5H₂O

Calculated %: C 59.41; H 5.48; N 6.93

Actual %: C 59.39; H 5.56; N 6.92

Examples 2 to 4

Compounds 2 to 4 were synthesized from cinnamic acids and correspondingamines according to the conditions described for the preparation ofcompound 1.

Ex- Mass am- spectrum ple R1 R2 R3 Compound name (M + H)⁺ 2 F H 4-F1-[4-(4-Fluoro-benzyl)-piperazin- 3431-yl]-3-(2-fluoro-phenyl)-propenone 3 Br H 4-F3-(2-Bromo-phenyl)-1-[4-(4-fluoro- 403 benzyl)-piperazin-1-yl]-propenone4 Cl H 4-Me 3-(2-Chloro-phenyl)-1-[4-(4-methyl- 355benzyl)-piperazin-1-yl]-propenone

Example 53-(2,6-Difluoro-phenyl)-1-[4-(4-fluoro-benzyl)-piperazin-1-yl]-propenone

Example 5A 4-[3-(2,6-Difluoro-phenyl)-acryloyl]-piperazine-1-carboxylicAcid tert-butyl Ester

3-(2,6-Difluoro-phenyl)-acryloyl chloride (3.0 g, 14.8 mmol) in solutionin dichloromethane (70 ml) in the presence of PS DIEA (4.07 q, 13.5mmol, 3.33 mmol/g) is treated with piperazine-1-carboxylic acidtert-butyl ester (2.3 g, 12.3 mmol) at room temperature. After 6 hoursof agitation the reaction mixture is filtered, taken up withdichloromethane and washed with 1 N soda and with water. The organicphase is dried on MgSO₄, filtered and evaporated to dryness. The syrupobtained is purified by silica column chromatography and eluted with a95/4.5/0.5 to 90/9.5/0.5 CH₂Cl₂/MeOH/NH₄OH mixture. Product 5A isisolated in the form of an off-white solid (3.87 g, 89%).

¹H NMR, DMSO-d₆ (ppm): 1.42 (s, 9H); 3.37 (broad s, 4H); 3.58 (broad s,4H); 7.22 (m, 2H); 7.50 (m, 1H).

Example 5B -3-(2,6-Difluoro-phenyl)-1-piperazin-1-yl-propenone

4-[3(2,6-Difluoro-phenyl)-acryloyl]-piperazine-1-carboxylic acidtert-butyl ester (3.87 g, 10.97 mmol) in solution in toluene (50 ml) istreated with trifluoroacetic acid (30 ml, 395 mmol) at room temperature.After 2 hours of agitation the reaction mixture is evaporated todryness, taken up with dichloromethane and washed with 1 N soda and thenwith water. The organic phase is dried on MgSO₄, filtered and evaporatedto dryness. The crude product is isolated for the following reaction(2.3 g, 88%).

Example 5-3-(2,6-Difluoro-phenyl)-1-[4-(4-fluoro-benzyl)-piperazin-1-yl]-propenone

Compound 5B (100 mg, 0.42 mmol) in solution in dichloromethane (5 ml) inthe presence of triethylamine (Et₃N) (0.088 ml, 0.63 mmol) is treatedwith 4-fluorobenzyl bromide (0.078 ml, 0.63 mmol) at room temperature.After 15 hours of agitation the reaction mixture is diluted withdichloromethane and washed with water. The organic phase is dried onMgSO₄, filtered and evaporated to dryness. The syrup obtained ispurified by silica column chromatography and eluted with a 100/0 to90/10 CH₂Cl₂/MeOH mixture. Product 5 is isolated in the form of alight-beige solid (72 mg, 48%).

Mass spectrum (ESI+): m/z 361 (M+H⁺)

Examples 6 to 12

Compounds 6 to 12 were synthesized from intermediate 5B andcorresponding benzyl chlorides or bromides according to the conditionsdescribed for the preparation of compound 5.

Ex- Mass am- spectrum ple R1 R2 Compound name (M + H)⁺ 6 4-Me H3-(2,6-Difluoro-phenyl)-1-[4-(4- 357 methyl-benzyl)-piperazin-1-yl]-propenone 7 3-Me 4-Me 3-(2,6-Difluoro-phenyl)-1-[4-(3,4- 371dimethyl-benzyl)-piperazin-1-yl]- propenone 8 3-F 4-F1-[4-(3,4-Difluoro-benzyl)-piperazin-1- 379yl]-3-(2,6-difluoro-phenyl)-propenone 9 4-Cl H1-[4-(4-Chloro-benzyl)-piperazin-1-yl]- 3773-(2,6-difluoro-phenyl)-propenone 10 3-Me H3-(2,6-Difluoro-phenyl)-1-[4-(3- 357 methyl-benzyl)-piperazin-1-yl]-propenone 11 3-Cl H 1-[4-(3-Chloro-benzyl)-piperazin-1-yl]- 3773-(2,6-difluoro-phenyl)-propenone 12 2-Me H3-(2,6-Difluoro-phenyl)-1-[4-(2- 357 methyl-benzyl)-piperazin-1-yl]-propenone

Examples 13 to 21 Example 13A-3-(2-chloro-phenyl)-1-piperazin-1-yl-propenone

Compound 13A was prepared in two steps from 3-(2-chloro-phenyl)-acryloylchloride according to the conditions described for the preparation ofcompound 5B.

Examples 13 to 21

Compounds 13 to 21 were synthesized from intermediate 13A according tothe conditions described for the preparation of compound 5.

Ex- Mass am- spectrum ple R1 R2 Compound name (M + H)⁺ 13 3-Me H3-(2-Chloro-phenyl)-1-[4-(3-methyl- 355benzyl)-piperazin-1-yl]-propenone 14 4-Cl H1-[4-(4-Chloro-benzyl)-piperazin-1- 375yl]-3-(2-chloro-phenyl)-propenone 15 2-F H3-(2-Chloro-phenyl)-1-[4-(2-fluoro- 359benzyl)-piperazin-1-yl-]-propenone 16 2-Me H3-(2-Chloro-phenyl)-1-[4-(2-methyl- 355benzyl)-piperazin-1-yl]-propenone 17 2-Cl H1-[4-(2-Chloro-benzyl)-piperazin-1- 375yl]-3-(2-chloro-phenyl)-propenone 18 3-F H3-(2-Chloro-phenyl)-1-[4-(3-fluoro- 359benzyl)-piperazin-1-yl]-propenone 19 3-Cl H1-[4-(3-Chloro-benzyl)-piperazin-1- 375yl]-3-(2-chloro-phenyl)-propenone 20 2-F 3-F3-(2-Chloro-phenyl)-1-[4-(2,3-difluoro- 377benzyl)-piperazin-1-yl]-propenone 21 3-F 4-F3-(2-Chloro-phenyl)-1-[4-(3,4-difluoro- 377benzyl)-piperazin-1-yl]-propenone

Example 22 3-(2,6-Difluoro-phenyl)-1[4-(2-fluoro-benzyl)-piperazin-1-yl]-propenone

A mixture of intermediate 5B (60 mg, 0.25 mmol) and2-fluoro-benzaldehyde (0.031 ml, 0.3 mmol) in solution indichloromethane (3 ml) in the presence of acetic acid (0.057 ml, 1.0mmol) is treated with MP-BH₃CN (117 mg, 0.275 mmol, 2.35 mmol/g) at roomtemperature. After 24 hours of agitation the reaction mixture isfiltered on a ChemElut cartridge previously impregnated with 1 N NaOHand then evaporated to dryness. The syrup obtained is purified by silicacolumn chromatography and eluted with a 100/0 to 95/5 CH₂Cl₂/MeOHmixture. Product 22 is isolated in the form of a yellow syrup (23 mg,25%).

Mass spectrum (ESI+): m/z 361 (M+H⁺)

Example 231-[4-(2-Fluoro-benzyl)-piperazin-1-yl]-3-(2-nitro-phenyl)-propenone

Example 23A 3-(2-Nitro-phenyl)-1-piperazin-1-yl-propenone

Compound 23A was prepared in two steps from 3-(2-nitro-phenyl)-acryloylchloride according to the conditions described for the preparation ofcompound 5B.

Example 23 1-[4-(2-Fluoro-benzyl)-piperazin-1-yl]3(2-nitro-phenyl)-propenone

Compound 23 was synthesized from compound 23A according to theconditions described for the preparation of compound 22.

Mass spectrum (ESI+): m/z 370 (M+H⁺)

Example 24 1-(4-Cyclohexylmethyl-piperazine-1-yl)-3-(2,6difluoro-phenyl)-propenone

Compound 24 was synthesized from compound 5B according to the conditionsdescribed for the preparation of compound 22.

Mass spectrum (ESI+): m/z 349 (M+H⁺)

Examples 25 to 28

Compounds 25 to 28 were synthesized from 1-cyclohexylmethyl-piperazineand corresponding cinnamic acids according to the conditions describedfor the preparation of compound 1.

Mass Exam- spectrum ple R1 R2 Compound name (M + H)⁺ 25 NO₂ H1-(4-Cyclohexylmethyl-piperazin-1-yl)- 358 3-(2-nitro-phenyl)-propenone26 CN H 2-[3-(4-Cyclohexylmethyl-piperazin-1- 338yl)-3-oxo-propenyl]-benzonitrile 27 F H1-(4-Cyclohexylmethyl-piperazin-1-yl)- 331 3-(2-fluoro-phenyl)-propenone28 Cl H 3-(2-Chloro-phenyl)-1-(4- cyclohexylmethyl-piperazin-1-y1)- 347propenone

Examples 29 to 33

Compounds 29 to 33 were synthesized from compound 23A and correspondingbenzyl chlorides or bromides according to the conditions described forthe preparation of compound 5.

Mass spectrum Example R1 R2 Compound name (M + H)⁺ 29 4-F H1-[4-(4-Fluoro-benzyl-piperazin-1- 370 yl]-3-(2-nitro-phenyl)-propenone30 4-Me H 1-[4-(4-Methyl-benzyl)-piperazin-1- 366yl]-3-(2-nitro-phenyl)-propenone 31 3-F 4-F1-[4-(3,4-Difluoro-benzyl)-piperazin- 3881-yl]-3-(2-nitro-phenyl)-propenone 32 4-Cl H1-[4-(4-Chloro-benzyl)-piperazin-1- 386 yl]-3-(2-nitro-phenyl)-propenone33 3-Me H 1-[4-(3-Methyl-benzyl)-piperazin- 3661-yl]-3-(2-nitro-phenyl)-propenone

Example 341-[4(2,6-Dimethyl-benzyl)-piperazin-1-yl]-3-(2-nitro-phenyl)-propenone

A mixture of compound 23A (70 mg, 0.27 mmol) and 2,5-dimethylbenzaldehyde (40 mg, 0.32 mmol) in solution in dichloroethane (3 ml) inthe presence of acetic acid (0.092 ml, 1.6 mmol) is treated withNaBH(OAc)₃ (63 mg, 0.297 mmol) at room temperature. After 24 hours ofagitation the reaction mixture is treated with saturated NaHCO₃ (2 ml),filtered on a ChemElut cartridge and evaporated to dryness.

The syrup obtained is purified by silica column chromatography andeluted with a 100/0 to 90/10 CH₂Cl₂/MeOH (+10% NH₄OH) mixture. Product34 is isolated and then salified by the addition of HCl in ether toyield a white solid (40 mg, 40%).

Mass spectrum (ESI+): m/z 380 (M+H⁺)

Examples 35 to 38

Compounds 35 to 38 were synthesized from compounds 5B, 13A and 23A andcorresponding phenethyl chlorides or bromides according to theconditions described for the preparation of compound 5.

Mass spectrum Example R1 R2 R3 Compound name (M + H)⁺ 35 F F H3-(2,6-Difluoro-phenyl)-1- 357 (4-phenethyl-piperazin-1- yl)-propenone36 NO₂ H H 3-(2-Nitro-phenyl)-1-(4- 366 phenethyl-piperazin-1-yl)-propenone 37 Cl H H 3-(2-Chloro-phenyl)-1-(4- 355phenethyl-piperazin-1-yl)- propenone 38 Cl H 4-F3-2-Chloro-phenyl)-1-{4-[2-(4- 373 fluoro-phenyl)-ethyl]-piperazin-1-yl}-propenone

The derivatives of the present invention are PAR-1 receptor antagonistsas the results of the models described below demonstrate:

In a variety of cell types, activation of PAR-1 receptors by the SFLLRpeptide (a selective PAR-1 agonist) triggers an intracellular signalcascade leading to the release of calcium by the endoplasmic reticulum.Chinese hamster ovarian (CHO) cells constituently express PAR-1receptor. In this cell line, the release of calcium consecutive toreceptor activation by SFLLR is measured by a fluorometry technique(fluorometric imaging plate reader, or FLIPR) using a selective probefor calcium (Fluo-3AM). The emission of fluorescence ispharmacologically proportional to the efficiency of the PAR-1 agonistand to its concentration. The compounds described in the presentinvention have demonstrated that they are capable of antagonizing PAR-1receptors and thus decreasing the release of calcium induced by theagonist.

Materials:

Culture medium: Ham's F-12 (Ham, R. G., Proc. Nat. Acad. Sci. 1965, 53:288) supplemented with 10% fetal calf serum and antibiotic (Probenicid,2.5 mM).

Fluorescent probe: Fluo-3AM (4 μM; Teflabs, Austin, Tex., USA)

Agonist: SFLLR NH₂ (Serine, phenylalanine, leucine, leucine, arginine).

Methods: CHO cells are inoculated in 96-well plates (60,000 cells perwell) in the presence of 200 μl of culture medium for 24 hours. Thecells are incubated with the calcium fluorescent probe for 1 hour at 37°C. The cells are then washed 10 minutes before the signal is measured.PAR-1 antagonist is then injected (0.01 μM to 10 μM). The plates areplaced in the FLIPR (Molecular Devices, UK) to measure calciumfluorescence at two wavelengths (488 nm and 540 nm: Sullivan et al.,Calcium Signaling Protocols 1999, 125-136). Measurements are taken for 5min before the antagonist is added and for 10 min following itsadministration. Maximum fluorescence minus baseline fluorescence ismeasured in 4 different wells. The test is carried out in duplicate.Under these conditions, the derivatives of the present invention wereidentified as PAR-1 receptor antagonists (antagonism >60% of the calciumsignal at 10 μM). The dose-response curves (0.01 μM to 32 μM) obtainedwith the SFLLR agonist allowed determination of the effectiveconcentration inducing 50% of the maximum effect (EC₅₀). The strengths(pA2) of some of the PAR-1 antagonists described in the presentinvention were calculated using the method of Arunlakshana and Schild(Brit. J. Pharmacol., 1959, 14: 48-58) from the EC₅₀ shifts observed atthree concentrations.

Results:

The several examples which follow, chosen among the compounds of thepresent invention, illustrate the completely unexpected capacity ofthese compounds to antagonize PAR-1 receptors.

Examples pA2 1 6.42 2 6.50 3 6.36 4 6.05 8 6.32 10 6.46 15 6.39 25 6.7828 6.12 29 6.98

The in vivo antiplatelet aggregation and antithrombotic activities ofPAR-1 antagonists have been shown in a guinea pig model of arterialthrombosis, which has very high hemodynamic shear stress. In a vascularbed, an endothelial lesion causes the intravascular formation of aplatelet-rich thrombus that will gradually occlude all of the vessel'slumen. The platelet aggregation process is strongly activated bythrombin via PAR-1 receptors. The compounds described in the presentinvention have demonstrated that they are capable of antagonizing PAR-1receptors and thus delaying thrombus formation.

Materials:

The studies are conducted using guinea pigs (PAR-1 receptors similar toman). Irradiation by means of a green laser light in the presence of aphotosensitizing agent (Rose Bengal administered intravenously) damagesthe carotid endothelium. The carotid flow rate is quantified using aTransonic flow probe. The time required to completely occlude thecarotid (flow rate of 0) is measured.

Methods:

After the animal is anesthetized (60 mg/kg pentobarbital), 5 mm of thecarotid artery is resected and the laser is placed 4 mm above theartery. A flow probe placed upstream measures occlusion time. RoseBengal (20 mg/kg) is administered by intravenous route and the vessel isirradiated at a wavelength of 514 nm (for 3 min). PAR-1 antagonists areadministered by intravenous route using a bolus (over 2 main immediatelybefore administration of Rose Bengal) followed by a 15-minute perfusionwhich begins when the laser is turned on.

Results:

Certain compounds described in the present invention have shown thatthey are able, after administration by intravenous route at doses from0.16 mg/kg to 2.5 mg/kg, to delay the time before the formation of athrombus from 5% to 135% compared to animals receiving vehicle alone.

The derivatives according to the invention are also of use in thetreatment of atrial fibrillation.

In the case of postinfarction cardiac cavity volume overload, the rightand left auricles dilate, thus constituting the substrate for thegenesis of atrial fibrillation. The disturbance of hemostasis in thecavity of the dilated auricle of a patient suffering from atrialfibrillation leads to an abnormal concentration of thrombin. Theinventors have demonstrated that this accumulation of thrombin isresponsible for an up-regulation of PAR-1 which can trigger theproliferation of fibroblasts as well as the formation of plateletthrombus.

By their mechanism of action, PAR-1 antagonists can thus prevent atrialdilation, fibroblast proliferation and thrombus formation in the auricleof a patient suffering from atrial fibrillation.

As a result, a PAR-1 antagonist constitutes an effective preventativeand/or curative treatment for atrial fibrillation. The compoundsdescribed in the present invention have demonstrated that they arecapable of antagonizing PAR-1 receptors and preventing auricle dilation.

Materials:

The studies are carried out using male rats. Because they toleratesurgery best, rats in a weight range of 180-200 g on arrival were chosenfor the experiment. Measurements of the various myocardial cavities areconducted by echocardiography on the anesthetized animal.

Methods:

The animal is anesthetized by a 3.5% mixture of isoflurane in oxygen(Aerrane, Baxter Laboratories). A thoracotomy perpendicular to thesternum of approximately 2 cm is performed at the level of the fourthintercostal space towards the left forefoot. A ligature (4-0 silk, CC1needle, Ethicon) is passed around the left coronary artery 1 mm from itsorigin. A surgical knot, sufficiently tight to completely occlude thevessel, is tied around the left coronary artery. Thecontinuously-recording electrocardiogram makes it possible to verify thesatisfactory positioning of the ligature. Two months after theprocedure, the animals are again anesthetized for an echocardiographicmeasurement of the cardiac cavities and a measurement of blood velocitywithin the myocardium using pulsed Doppler. Lastly, the animals areeuthanized by sodium pentobarbital overdose (160 mg/kg, IP) for varioushistological measurements. The animals are force-fed daily PAR-1antagonist products from 24 h after infarction until the animal issacrificed.

Results:

Certain compounds described in the present invention have shown thatthey are able, after administration by oral route in doses from 10-100mg/kg/d for 60 days, to reduce by 20% to 90% the auricle surface(measured by echocardiography) compared to untreated animals.

The present invention also relates to pharmaceutical compositionscontaining as an active ingredient a compound of general formula (I), ora pharmaceutically-acceptable salt thereof, mixed or combined with asuitable excipient. Such compositions can assume the form, for example,of solid or liquid compositions, emulsions, lotions or creams.

As solid compositions for oral administration, tablets, pills, powders(in gelatin capsules or in packets) or granules can be used. In suchcompositions, the active ingredient according to the invention is mixedwith one or more inert diluents, such as starch, cellulose, sucrose,lactose or silica, under an argon flow. Such compositions may alsoinclude substances other than diluents, for example one or morelubricants such as magnesium stearate or talc, a colorant, a coating(for sugar-coated pills) or a varnish.

As liquid compositions for oral administration, the following can beused: pharmaceutically-acceptable solutions, suspensions, emulsions,syrups and elixirs containing inert diluents such as water, ethanol,glycerol, vegetable oils or liquid paraffin. Such compositions caninclude substances other than diluents, for example wetting, sweetening,thickening, flavoring or stabilizing agents.

Sterile compositions for parenteral administration can be, preferably,aqueous or non-aqueous solutions, suspensions or emulsions. As a solventor vehicle, the following can be used: water, propylene glycol,polyethylene glycol, vegetable oils, in particular olive oil, injectableorganic esters, for example ethyl oleate or other suitable organicsolvents. Such compositions can also contain additives, in particularwetting agents, isotonic agents, emulsifiers, dispersants andstabilizers. Sterilization can be achieved in several ways, for exampleby sterilizing filtration, by incorporating sterilizing agents in thecomposition, by irradiation or by heating. Such compositions can also beprepared in the form of sterile solid compositions that can be dissolvedin sterile water or in any other injectable sterile medium just beforeuse.

Compositions for rectal administration are suppositories or rectalcapsules that contain, in addition to the active product, excipientssuch as cocoa butter, semi-synthetic glycerides or polyethylene glycols.

Compositions for topical administration can be creams, lotions, eyedrops, mouth washes, nose drops or aerosols, for example.

Doses depend on desired effect, treatment duration and administrationroute, and are generally between 0.001 g and 1 g (preferably between0.005 g and 0.75 g) per day, preferably by oral route for an adult, withunit doses ranging from 0.1 mg to 500 mg of active substance.

Generally, the doctor will establish suitable dosing according to thepatient's age, weight and other specific factors of the case.

According to a specific embodiment, the present invention also relatesto products containing a compound according to general formula (I) andanother cardiovascular agent as a combination product for simultaneous,separate or time-release use in cardiovascular therapy, the othercardiovascular agent able to be an antiplatelet agent such as aspirin,clopidogrel, ticlopidine, abciximab, tirofiban or eptifibatide.

According to additional characteristics of the present invention,compounds of general formula (I) are of use in the manufacture of a drugto inhibit the proliferation of smooth muscle cells (restenosis) and/orfor the curative and/or preventive treatment of the proliferation ofendothelial, fibroblast, cardiofibroblast, glial, smooth muscle orcancer cells.

1. Compounds of general formula (I):

wherein: R₁ represents: halogen, CN or NO₂; R₂ represents: hydrogen orhalogen; n represents; 1 or 2; R₃ represents: phenyl substituted by oneor more halogens or C₁-C₆ alkyls; or a cyclohexyl; as well as thetherapeutically-acceptable salts or solvates thereof.
 2. Compoundsaccording to claim 1, wherein R₁ is halogen, R₂ is hydrogen, n equals 1and R₃ is phenyl substituted by one or more halogens or C₁-C₆ alkyls. 3.Compounds according to claim 1, wherein R₁ is cyano, R₂ is hydrogen, nequals 1 and R₃ is phenyl substituted by one or more halogens or C₁-C₆alkyls.
 4. Compounds according to claim 1, wherein R₁ is halogen, R₂ ishydrogen, n equals 1 and R₃ is cyclohexyl.
 5. Compounds according toclaim 1, wherein R₁ is cyano, R₂ is hydrogen, n equals 1 and R₃ iscyclohexyl.
 6. A compound according to claim 1 selected among:3-(2-Chloro-phenyl)-1-[4-(4-fluoro-benzyl)-piperazin-1-yl]-propenone;1-[4-(4-Fluoro-benzyl)-piperazin 1-yl]-3-(2-fluoro-phenyl)-propenone;3-(2-Bromo-phenyl)-1-[4-(4-fluoro-benzyl)-piperazin-1-yl]-propenone;3-(2-Chloro-phenyl) 1-[4-(4-methyl-benzyl)-piperazin-1-yl]-propenone;3-(2,6-Difluoro-phenyl)-1-[4-(4-fluoro-benzyl)-piperazin-1-yl]-propenone;3-(2,6-Difluoro-phenyl)-1-[4-(4-methyl-benzyl)-piperazin-1-yl]-propenone;3-(2,6-Difluoro-phenyl)-1-[4-(3,4-dimethyl-benzyl)-piperazin-1-yl]-propenone;1-[4-(3,4-Difluoro-benzyl)-piperazin1-yl]-3-(2,6-difluoro-phenyl)-propenone;1-[4-(4-Chloro-benzyl)-piperazin-1-yl]-3-(2,6-difluoro-phenyl)-propenone;3-(2,6Difluoro-phenyl)-1-[4-(3-methyl-benzyl)-piperazin-1-yl]-propenone;1-[4-(3-Chloro-benzyl)-piperazin-1-yl]-3-(2,6-difluoro-phenyl)-propenone;3-(2,6-Difluoro-phenyl)-1-[4-(2-methyl-benzyl)-piperazin-1-yl]-propenone;3-(2-Chloro-phenyl)-1-[4-(3-methyl-benzyl)-piperazin-1-yl]-propenone;1-[4-(4-Chloro-benzyl)-piperazin-1-yl]-3-(2-chloro-phenyl)-propenone;3-(2-Chloro-phenyl)-1-[4-(2-fluoro-benzyl)-piperazin-1-yl]-propenone;3-(2-Chloro-phenyl)-1-[4-(2-methyl-benzyl)-piperazin-1-yl]-propenone;1-[4(2-Chloro-benzyl)-piperazin-1-yl]3-(2-chloro-phenyl)-propenone;3-(2-Chloro-phenyl)-1-[4-(3-fluoro-benzyl)-piperazin-1-yl]-propenone;1-[4(3-Chloro-benzyl)-piperazin-1-yl]-3-(2-chloro-phenyl)-propenone;3-(2-Chloro-phenyl)-1-[4-(2,3-difluoro-benzyl)-piperazin-1-yl]-propenone;3-(2-Chloro-phenyl)-[4-(3,4-difluoro-benzyl)-piperazin-1-yl]-propenone;3-(2,6-Difluoro-phenyl)-1-[4-(2-fluoro-benzyl)-piperazin-1-yl]-propenone;1-[4-(2 Fluoro-benzyl)-piperazin-1-yl]-3-(2-nitro-phenyl)-propenone;1-(4-Cyclohexylmethyl-piperazin-1-yl)-3-(2,6 difluoro-phenyl)-propenone;2-[3-(4-Cyclohexylmethyl-piperazin-1-yl)-3-oxo-propenyl]-benzonitrile;1-(4Cyclohexylmethyl-piperazin-1-yl)-3-(2 nitro phenyl)-propenone;1-(4Cyclohexylmethyl-piperazin-1-yl)-3-(2-fluoro-phenyl)-propenone; 3-(2Chloro-phenyl)-1-(4-cyclohexylmethyl-piperazin-1-yl)-propenone;1-[4-(4-Fluoro benzyl)-piperazin 1-yl]-3-(2-nitro-phenyl)-propenone;1-[4-(4-Methyl-benzyl)-piperazin-1-yl]-3-(2-nitro-phenyl)-propenone;1-[4-(3,4-Difluoro-benzyl)-piperazin-1-yl]-3-(2-nitro-phenyl)-propenone;1-[4-(4-Chloro-benzyl)-piperazin-1-yl]-3-(2-nitro-phenyl)-propenone;1-[4-(3-Methyl-benzyl)-piperazin-1-yl]-3-(2-nitro-phenyl)-propenone;1-[4-(2,6-Dimethyl-benzyl)-piperazin-1-yl]-3-(2-nitro-phenyl)-propenone;3-(2,6-Difluoro-phenyl)-1-(4-phenethyl-piperazin-1-yl)-propenone;3-(2-Chloro-phenyl)-1-(4-phenethyl-piperazin-1-yl)-propenone;3-(2-Chloro-phenyl)-1-{4-[2-(4-fluoro-phenyl)-ethyl]-piperazin-1-yl}-propenone;as well as therapeutically-acceptable salts and solvates thereof. 7.Compounds according to claim 1, for use as a drug.
 8. A method ofpreparation of compounds of general formula (I) according to claim 1,comprised of the condensation of an intermediate of general formula (II)

wherein R₁ and R₂ are defined as in the description of general formula(I) of claim 1, X can represent a leaving group such as chlorine or Xcan represent hydroxyl, with an amine of general formula (III)

wherein P₁ represents a protective group. The intermediate obtained, ofgeneral formula (IV)

wherein R₁, R₂ and P₁ are defined as previously, yields compounds ofgeneral formula (I) after deprotection and reaction of the amineachieved either with a reagent of general formula R₃(CH₂)_(n)Y whereinR₃ is defined as previously and Y represents a leaving group such as,for example, Cl, Br, I, OSO₂CH₃, OSO₂CF₃ or O-tosyl, or with an aldehydeof formula R₃—(CH₂)_(n-1)—CHO wherein R₃ and n are defined aspreviously.
 9. A method of preparation of compounds of general formula(I) according to claim 1, comprised of the condensation of anintermediate of general formula (II)

wherein R₁, R₂ and X are defined as previously with all amine of generalformula (V)

wherein n and R₃ are defined as previously yielding to compounds ofgeneral formula (I).
 10. Pharmaceutical compositions containing as anactive ingredient at least one compound according to claim 1, incombination with a pharmaceutically-acceptable vehicle.
 11. The use of acompound according to claim 1 for the manufacture of a thrombin-receptorantagonist drug.
 12. The use of a compound according to claim 1 for themanufacture of a drug for the curative and/or preventive treatment ofdisorders related to PAR-1 (protease-activated receptor-1) activation.13. The use of a compound according to claim 1 for the manufacture of anantiplatelet aggregation drug.
 14. The use of a compound according toclaim 1 for the manufacture of a drug for the curative and/or preventivetreatment of arterial and/or venous thrombosis.
 15. The use of acompound according to claim 1 for the manufacture of a drug for thecurative and/or preventive treatment of stable angina, heart rhythmdisorders, cerebral vascular accidents, heart failure, hypertension ormyocardial infarction.
 16. The use of a compound according to claim 1for the manufacture of a drug for the curative and/or preventivetreatment of atrial fibrillation and myocardial remodeling.
 17. The useof a compound according to claim 1 for the manufacture of a drug for thecurative and/or preventive treatment of acute coronary syndromes. 18.The use of a compound according to claim 1 for the manufacture of a drugto inhibit the proliferation of smooth muscle cells (restenosis). 19.The use of a compound according to claim 1 for the manufacture of a drugfor the curative and/or preventive treatment of inflammatory disorderspulmonary diseases, gastrointestinal diseases, fibrosis development inchronic liver disease patients or skin diseases.
 20. The use of acompound according to claim 1 for the manufacture of a drug for thecurative and/or preventive treatment of the proliferation ofendothelial, fibroblast, cardiofibroblast, glial, smooth muscle orcancer cells.
 21. A product containing at least one compound accordingto claim 1 and another cardiovascular agent as a combination product forsimultaneous, separate or time-release use in cardiovascular therapy.22. A product according to claim 20, wherein the other cardiovascularagent is an antiplatelet aggregation agent such as aspirin, clopidogrel,ticlopidine, abciximab, tirofiban or eptifibatide.
 23. The use of anantagonist PAR 1 for the manufacture of a drug for the curative and/orpreventive treatment of atrial fibrillation.